The innate immune system is the first line of defense against pathogens. Innate immune cells lack the exquisite specificity of the adaptive immune system, yet in order to respond in a measured way they must be able to tailor their response to the specific pathogen. These cells have therefore evolved pattern recognition receptors including the Toll-like receptors (TLRs) that recognize conserved molecular patterns characteristic of the microbe, which are not found within the host. While much is known about the mechanisms through which TLRs mediate immune responses, a number of critical questions remain unanswered. Central to these is a complete knowledge of all the components of the TLR signaling pathways and an understanding of how the architectural arrangement of these components leads to the appropriate coordination of host defense. We have utilized the tools of systems biology to discover Sharpin, a novel component of TLR signaling, and our preliminary studies of the protein have altered our understanding of the architecture of the TLR pathway. We have demonstrated that Sharpin acts at the level of the NEMO-containing IKK complex and controls a novel branch point in the TLR2/NF-?B pathway that is necessary for the production of Th1 cytokines. In this proposal, we will: 1. Determine the mechanism by which Sharpin influences transcriptional responses of TLR- dependent genes through epigenetic modification of chromatin structure and enhanceosome formation. 2. Delineate the manner by which Sharpin transduces TLR2/MyD88 signals through the IKK complex to regulate transcriptional responses. 3. Determine the role of Sharpin in controlling innate and adaptive immune responses to bacterial pathogens in vivo. The proposed study will advance our knowledge of the TLR2/MyD88 pathway, NF-?B activation, epigenetic regulation of innate immune genes, and mechanisms underlying the instruction of adaptive responses by innate immune cells.